Structure optimization and preliminary bioactivity evaluation of N-hydroxybenzamide-based HDAC inhibitors with Y-shaped cap

Histone deacetylase inhibitors (HDACIs) are effective small molecules in the treatment of human cancers. In our continuing efforts to develop novel N-hydroxyterephthalamide-based HDACIs, herein we report the design and development of a new class of N-hydroxybenzamide-based HDACIs. In this new class of analogs, we inserted an ethylene moiety in the linker and used indole as a part of the Y-shaped cap group. Biological characterization identified compounds 4o, 4p, 4q and 4t to show improved HDAC inhibition, while no isoform selectivity for HDACs was observed. These compounds also exhibited improved anti-proliferative activity against multiple cancer cell lines when compared to their parent compound and positive control SAHA.     

Inhibition of tryptophanyl-tRNA synthetase by modifying ATP analogs

The interaction between tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef pancreas and the ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of ATP was studied as an approach to investigate the structure of the enzyme active center. Some of the compounds under study were shown to irreversibly inhibit the enzyme activity; the presence of ATP in the most cases protects the enzyme against inactivation. The kinetic constants Ki and k2 of interaction of the irreversible inhibitors with the enzyme were determined. It was found that the Ki values for a number of irreversible competitive inhibitors are by 1-2 orders of magnitude less than the Km value for ATP; the k2 values were found equal to 0.02-0.04 min-1. this suggests that the compounds may be used as affinity reagents, the most efficient ones being adenosine 5'-(beta-chloroethyl phosphate) and mixed AMP-mesithylene carbonic acid anhydride. The absence of a protective effect of ATP in the case of adenosine 5'-(beta-bromoethane phosphonate) and non-competitive type of reversible inhibition inhibition of the enzyme by adenosine 5'-chloromethane phosphonate indicate that the molecule of tryptophanyl-tRNA synthetase contains sites interacting with adenine nucleotides, other than the ATP binding sites of the active center.     

Monoamine oxidase inhibition by C4-substituted phthalonitriles

It was recently reported that a series of C5-substituted phthalimides are remarkably potent reversible inhibitors of recombinant human monoamine oxidase (MAO) B. Modeling studies suggested that the phthalimide ring forms numerous polar interactions with the polar region of the MAO-B substrate cavity while the C5 side chain extends to, and interacts via Van der Waals interactions with the hydrophobic regions of the enzyme entrance cavity. Interactions with both cavities appear to be requirements for high affinity binding. In the present study we have examined an analogs series of C4-substituted phthalonitriles as potential human MAO inhibitors. The phthalonitriles were found to be highly potent reversible MAO-B inhibitors with most analogs exhibiting IC(50) values in the low nM range. The phthalonitriles also interacted with human MAO-A, although with lower binding affinities compared to MAO-B. Modeling studies suggest that the high binding affinities of the phthalonitriles to MAO-B may depend, at least in part, on the formation of polar interactions between the nitrile functional groups and the enzyme substrate cavity. Examination of a homologs series of benzonitriles established that the phthalonitrile moiety is more optimal for MAO-B inhibition than the corresponding benzonitrile moiety, and that C3-substituted benzonitriles are better MAO-B inhibitors than C4-substituted benzonitriles. Since elimination of the nitrile functional group yielded compounds with only moderate MAO-B inhibition potencies, it may be concluded that this functional group is privileged for MAO-B inhibition.     

Capsaicin and its analogs inhibit the activity of NADH-coenzyme Q oxidoreductase of the mitochondrial respiratory chain

Capsaicin and its analogs with different acyl moieties were found to inhibit the electron-transfer activity of NADH-coenzyme Q oxidoreductase isolated from beef heart mitochondria. The inhibitory potency of capsaicin was lower than those of dihydrocapsaicin and analogs with heptanoyl, capryl, undecanoyl, and lauroyl moieties, but was higher than those of analogs with palmitoyl and stearoyl moieties. The analog with the lauroyl moiety showed the strongest inhibition. These results suggest that hydrophobicity and the appropriate carbon chain length of the acyl moiety are important for the binding of compounds to the enzyme. On the other hand, capsaicin and its analogs did not interrupt rotenone-insensitive electron transfer from NADH to menadione. Furthermore, these compounds had almost no effect on the spectral properties and EPR signals arising from iron-sulfur clusters of the NADH-treated enzyme. Kinetic analyses with double-reciprocal plots showed that these compounds were competitive inhibitors with respect to coenzyme Q1, an electron acceptor. These results strongly suggest that capsaicin and its analogs bind to the coenzyme Q1 binding site of the enzyme.     

Sulfonyl-morpholino-pyrimidines: SAR and development of a novel class of selective mTOR kinase inhibitor

High throughput screening to identify inhibitors of the mTOR kinase revealed sulfonyl-morpholino-pyrimidine 1 as an attractive start point. The compound displayed good physicochemical properties and selectivity over related kinases such as PI3Kα. Library preparation of related analogs allowed the establishment of additional SAR understanding and in particular the requirement for a key hydrogen bond donor motif at the 4-position of the phenyl ring in compounds such as indole 19. Isosteric replacement of the indole functionality led to the identification of urea compounds such as 32 that show good levels of mTOR inhibition in both enzyme and cellular assays.     

Characterization of two types of binding sites of substrate-like inhibitors in the heavy meromyosin molecule

The effects of several phosphorylating and alkylating analogs of the substrate on the ATPase activity of myosin and heavy meromyosin were compared. The data obtained confirmed the previously made assumption on the existence of two types of substrate-like inhibitor binding sites in the enzyme molecule. In one of the sites, presumably in the active one, there occurs a reversible competitive inhibition characterized by a high affinity for the inhibitors, which are mixed anhydrides of various mononucleotides and mesitylcarboxylic acid or its derivatives. An enhancement of hydrophobicity of these compounds causes an increase in their affinity for this site. At much higher concentrations of the inhibitors an irreversible inhibition takes place, the rate of inhibition being decreased with an increase in the phosphorylating capacity of the compound. This site possesses a far lower affinity for the inhibitors and reveals a certain specificity with respect to the analog mononucleotide moiety structure, i.e. a replacement of the 6-NH2-group by the 6-OH-group or an increase in the number of the phosphate residues result in a decrease of the efficiency of inhibition. No correlation between the analog capacity to cause irreversible inhibition and to act as an effective competitive inhibitor of reversible type has been shown to exist, thus allowing to use inhibitors of preferable action in one of the two types of the binding sites. No irreversible inhibition site was revealed when the ATPase activity of myosin subfragment I with and without the DTNB chains was investigated. Actin protects myosin against the inhibiting action of the analogs tested.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo)triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM)

The chorismate mutase (CM) is considered as an attractive target for the identification of potential antitubercular agents due to its absence in animals but not in bacteria. A series of 3-indolylmethyl substituted pyrazolotriazinone derivatives were designed and docked into CM in silico as potential inhibitors. These compounds were efficiently synthesized using the Pd/Cu-catalyzed coupling-cyclization in a single pot involving the construction of indole ring. The methodology was later extended to the preparation of corresponding benzo analogs of pyrazolotriazinones i.e. 3-indolylmethyl substituted benzotriazinone derivatives. Several of these novel compounds showed significant inhibition of CM when tested in vitro at 30 µM. The SAR (Structure-Activity-Relationship) studies suggested that benzotriazinone moiety was more favorable over the pyrazolotriazinone ring. The two best active compounds showed IC₅₀ ∼ 0.4–0.9 µM (better than the reference/known compounds used) and no toxicity till 30 µM in vitro.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INHIBITION STUDIES WITH N-(5′-PHOSPHOPYRIDOXYL) AMINO ACIDS1

Abstract— Seven N-(5′-phosphopyridoxyl) amino acids, reduced analogs of the glutamate-pyridoxal phosphate Schiff base, were synthesized and purified. All of them inhibited mouse brain glutamate decarboxylase activity. The four most potent inhibitors were the aminooxyacetate, GABA, cysteinesul-finate and glutamate derivatives, and the effect of these compounds was studied kinetically. The inhibition produced was in all cases mixed function with respect to glutamate and competitive with respect to pyridoxal phosphate. The inhibition kinetics were non-linear. These results are interpreted in terms of an ordered binding of pyridoxal phosphate and glutamate to the enzyme. Furthermore, they are consistent with previous findings suggesting the existence of two kinds of glutamate decarboxylase activity differing in their dependence on free pyridoxal phosphate.     

Kinetic and crystallographic studies on 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of beta-D-glucopyranose, 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K(i) values of 145.2 (+/-11.6), 76 (+/-4.8), and 8.6 (+/-0.7) muM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 A; GPb-benzothiazole, 2.10 A; GPb-benzimidazole, 1.93 A). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (approximately 32 A) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.     

Biochemical requirements for inhibition of Connexin26-containing channels by natural and synthetic taurine analogs

Previous work has shown that protonated taurine and aminosulfonate pH buffers, including HEPES, can directly and reversibly inhibit connexin channels that contain connexin26 (Cx26) (Bevans, C. G., and Harris, A. L. (1999) J. Biol. Chem. 274, 3711-3719). The structural requirements for this inhibition were explored by studies of the effects of structural analogs of taurine on the activity of Cx26-containing reconstituted hemichannels from native tissue. Several analogs inhibited the channels, with a range of relative affinities and efficacies. Each active compound contains a protonated amine separated from an ionized sulfonate or sulfinate moiety by several methylene groups. The inhibition is eliminated if the sulfonate/sulfinate moiety or the amine is not present. Compounds that contain a protonated amine but lack a sulfonate/sulfinate moiety do not inhibit but do competitively block the effect of the active compounds. Compounds that lack the protonated amine do not significantly inhibit or antagonize inhibition. The results suggest involvement of the protonated amine in binding and of the ionized sulfur-containing moiety in effecting the inhibition. The maximal effect of the inhibitory compounds is enhanced when a carboxyl group is linked to the alpha-carbon. Inhibition but not binding is stereospecific, with l-isomers being inhibitory and the corresponding d-isomers being inactive but able to antagonize inhibition by the l-isomers. Whereas not all connexins are sensitive to aminosulfonates, the well defined structural requirements described here argue strongly for a highly specific regulatory interaction with some connexins. The finding that cytoplasmic aminosulfonates inhibit connexin channels whereas other cytoplasmic compounds antagonize the inhibition suggests that gap junction channels are regulated by a complex interplay of cytoplasmic ligands.     

Inhibition of leucyl-tRNA-synthetases by modified ATP analogs

The interaction between modifying ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of the ATP molecule and leucyl-tRNA synthetases from cytoplasm and chloroplasts of Euglena gracilis (strain Z) was studied. It was shown that most of the ATP analogs irreversibly inhibit the cytoplasmic enzyme, having no inhibiting effect on the chloroplast synthetase. The kinetic constants K1 and k2 for the interaction between the most effective irreversible inhibitors and the cytoplasmic enzyme were determined. The data on the protection of the enzyme activity by substrates against irreversible inhibition suggest, that the effect of the adenosine 5'-(beta-chloroethyl phosphate) is directed to the ATP-binding site of the cytoplasmic enzyme, whereas the mixed anhydride of AMP and mesithylene carbonic acid acts predominantly on the binding site of 3'-terminal adenosine of the tRNALeu molecule. ATP analogs may be effectively used for affinity labelling of the cytoplasmic leucyl-tRNA synthetase.     

Inhibition of crayfish glutamic acid decarboxylase by structural analogs of the substrate and product

Crayfish glutamic acid decarboxylase (GAD) is inhibited by some aliphatic carboxylic acid analogs of glutamate and gamma-amino-n-butyric acid (GABA). Variations in the length of the carbon skeleton, substitution of a keto for a methylene group, replacement of the carboxyl group or attachment of a bulky basic moiety to the amino terminus of GABA all lead to a drastic reduction in its inhibitory activity. Substitution of a methyl group for the amino group of GABA is a permissible alteration which does not reduce the inhibitory potency. Some structural analogs of glutamate are inhibitory also, particularly if they possess a comparable carbon skeleton and a keto group in the alpha position or a sulfhydryl group. Most of the sulfhydryl analogs are significantly more potent as inhibitors than the corresponding compounds in which the SH group is replaced by an H atom.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Carbonic anhydrase inhibitors: synthesis and inhibition studies against mammalian isoforms I-XV with a series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides

A series of 2-(hydrazinocarbonyl)-3-substitutedphenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy- functionalities, as well as the perfluorophenyl moiety have been synthesized and evaluated as inhibitors of 13 catalytically active, mammalian carbonic anhydrase (CA, EC 4.2.1.1) isoforms, that is, CA I-CA XV (of human (h) or murine (m) origin). The new compounds were ineffective inhibitors of isozymes hCA III, hCA IV, hCA VA, hCA VB, hCA VI and mCA XIII, moderate inhibitors of hCA I, hCA VII, hCA IX and mCA XV, and excellent, low-nanomolar inhibitors of hCA II and hCA XIV. The substitution pattern of the aromatic group in the 3-position of the indole ring influenced biological activity and isozyme inhibition profiles in this series of sulfonamides. Some of the best and most selective hCA XIV and mCA XV inhibitors ever reported have been identified in this study.     

Novel trifluoroacetophenone derivatives as malonyl-CoA decarboxylase inhibitors

A series of trifluoroacetophenone derivatives were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Some of the 'reverse amide' analogs were found to be potent inhibitors of MCD enzyme activity. The trifluoroacetyl group may interact with the MCD active site as the hydrate in a similar fashion to the hexafluoroisopropanol analogs reported previously. Adding electron-withdrawing groups to the phenyl ring stabilizes the hydrated species and enhances this interaction.     

Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.     

Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: In vitro and in vivo studies

Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC(50) ranging from 1 to 10 microM, in the presence of 750 microM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K(I)=6 microM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 microM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme.     

Halogen-substituted (C-β-D-glucopyranosyl)-hydroquinone regioisomers: synthesis, enzymatic evaluation and their binding to glycogen phosphorylase

Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) 1,4-dimethoxybenzene (1) afforded regioselectively products halogenated at the para position to the D-glucosyl moiety (8, 9) that were deacetylated to 3 (chloride) and 16 (bromide). For preparing meta regioisomers, 1 was efficiently oxidized with CAN to afford C-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) 1,4-benzoquinone 2 which, in either MeOH or H(2)O-THF containing few equivalents of AcCl, added hydrochloric acid to produce predominantly meta (with respect to the sugar moiety) chlorinated hydroquinone derivatives 5 and 18, this latter being deacetylated to 4. The deacetylated meta (4, 5) or para (3, 16) halohydroquinones were evaluated as inhibitors of glycogen phosphorylase (GP, a molecular target for inhibition of hepatic glycogenolysis under high glucose concentrations) by kinetics and X-ray crystallography. These compounds are competitive inhibitors of GPb with respect to α-D-glucose-1-phosphate. The measured IC(50) values (μM) [169.9±10.0 (3), 95 (4), 39.8±0.3 (5) 136.4±4.9 (16)] showed that the meta halogenated inhibitors (4, 5) are more potent than their para analogs (3, 16). The crystal structures of GPb in complex with these compounds at high resolution (1.97-2.05 ?) revealed that the inhibitors are accommodated at the catalytic site and stabilize the T conformation of the enzyme. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions with protein residues of the different substituents on the aromatic part of the inhibitors.     

A new series of N2-substituted-5-(p-toluenesulfonylamino)phthalimide analogues as α-glucosidase inhibitors

Several members of a new family of non-sugar-type α-glycosidase inhibitors, bearing a 5-(p-toluenesulfonylamino)phthalimide moiety and various substituent at the N2 position, were synthesized and their activities were investigated. The newly synthesized compounds displayed different inhibition profile towards yeast α-glycosidase and rat intestinal α-glycosidase. Almost all the compounds had strong inhibitory activities against yeast α-glycosidase. Regarding rat intestinal α-glycosidase, only analogs with N2-aromatic substituents displayed varying degrees of inhibitory activities on rat intestinal maltase and lactase and nearly all compounds showed no inhibition against rat intestinal α-amylase. Structure–activity relationship studies indicated that 5-(p-toluenesulfonylamino)phthalimide moiety is a favorable scaffold to exert the α-glucosidase inhibitory activity and substituents at the N2 position have considerable influence on the efficacy of the inhibition activities.